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H Glossman

Publications and source records attributed to H Glossman.

6 recordsLinked to original sources
PubMed1996

Transfer of L-type calcium channel IVS6 segment increases phenylalkylamine sensitivity of alpha1A.

Conditioned ("use-dependent") inhibition by phenylalkylamines (PAAs) is a characteristic property of L- type calcium (Ca2+) channels. To determine the structural elements of the PAA binding domain we transferred sequence stretches of the pore-forming regions of repeat III and/or IV from the skeletal muscle alpha1 subunit (alpha1S) to the class A alpha1 subunit (alpha1A and expressed these chimeras together with beta1a and alpha2/delta subunits in Xenopus oocytes. The corresponding barium currents (IBa) were tested for PAA sensitivity during trains of depolarizing test pulses (conditioned block). IBa of oocytes expressing the alpha1A subunit were only weakly inhibited by PAAs (less than 10% conditioned block of IBa during a 100-ms pulse train of 0.1 Hz). Transfer of the transmembrane segment IVS6 from alpha1S to alpha1A produced an enhancement of PAA sensitivity of the resulting alpha1A/alpha1S chimera comparable to L-type alpha1 subunits (about 35% conditioned block Of IBa during a 100-ms pulse train of 0.1 Hz). Our results demonstrate that substitution of 11 amino acids within the segment RVS6 of alpha1A with the corresponding residues of alpha1S is sufficient to transfer L-type PAA sensitivity into the low sensitive class A Ca2+ channel.

Amino Acid Sequence↗
PubMed1992

In vivo labeling of L-type Ca2+ channels by fluorescent dihydropyridines: evidence for a functional, extracellular heparin-binding site.

We have synthesized and characterized fluorescently labeled dihydropyridines (DHPs) as probes for L-type Ca2+ channels. Racemic as well as (+)- and (-)-1,4-dihydro- 2,6-dimethyl-4-(2-trifluoromethylphenyl)-3,5-pyridinecarboxylic acid 2-(aminoethyl)ethyl ester hydrochlorides were coupled to boron dipyrromethane (Bodipy) derivatives. (4,4-Difluoro-5,7-dimethyl-4-bora-3a,4a-diaza)-3- (s-indacene)propionic acid (DMBodipy)-DHP and (4,4-difluoro-7-styryl-4-bora-3a,4a-diaza)-3-(s-indacene+ ++)propionic acid (STBodipy)-DHP have Kd values in the nanomolar range for membrane-bound or partially purified skeletal muscle and for neuronal L-type Ca2+ channels. (-)- and (+)-STBodipy-DHPs block 45Ca2+ uptake through L-type Ca2+ channels into GH3 cells with IC50 values of 14.8 and 562 nM, respectively. The measurement of bound fluorescence after removal of free DMBodipy-DHP with charcoal shows that the probes can substitute for radioactive ligands to study the properties (equilibrium binding, kinetics, allosteric regulation) of partially purified L-type Ca2+ channels from skeletal muscle. L-type Ca2+ channels on GH3 cells were steroselectively visualized by using the optical enantiomers of STBodipy-DHP. Heparin inhibited GH3 cell labeling by (-)-STBodipy-DHP with an IC50 value of 9.7 micrograms/ml and blocked L-type Ca(2+)-channel-mediated 45Ca2+ uptake with an IC50 value of 32 micrograms/ml. These findings argue for an extracellular orientation of the heparin-binding domain of the Ca2+ channel that is coupled to the DHP receptor.

Animals↗
PubMed1989

Stereoselective binding of niguldipine enantiomers to alpha 1A-adrenoceptors labeled with [3H]5-methyl-urapidil.

[3H]5-Methyl-urapidil, a potent antihypertensive derivative of urapidil, binds to alpha 1A-adrenoceptors in rat brain cortex membranes with a dissociation constant (KD) of 0.89 nM and a Bmax of 116 fmol/mg protein. The ligand does not bind to purified liver cell membranes (alpha 1B-adrenoceptors). [3H]5-Methyl-urapidil also labels 5-HT1A receptors in brain membranes (KD: 0.84 nM and Bmax: 235 fmol/mg protein). (+/-)-Niguldipine, a novel 1,4-dihydropyridine with Ca2+-antagonistic as well as alpha 1A-adrenoceptor blocking properties, is a competitive inhibitor of [3H]5-methyl-urapidil binding to alpha 1A-adrenoceptors. In contrast to those for prazosin, the Ki values for niguldipine were highly dependent on the membrane protein concentration, indicating partitioning of niguldipine into hydrophobic compartments unavailable for alpha-adrenoceptor interaction. The extrapolated, 'true' Ki values were as follows: (+/-)-niguldipine: 0.298 nM, (-)-niguldipine: 3.12 nM, (+)-niguldipine: 0.145 nM.

Animals↗
PubMed1981

[3H]-Nitrendipine, a potent calcium antagonist, binds with high affinity to cardiac membranes.

The tritiated calcium antagonist 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridine carboxylic acid, 3-ethyl-5-methyl ester (nitrendipine, Bay e 5009), a potent analogue of nifedipine, binds in a reversible and saturable manner to partially purified guinea-pig heart membranes. The analyses of the equilibrium binding data with Scatchard plots is in accord with either negative cooperativity of binding or with the assumption of two classes of binding sites, where one site has an equilibrium dissociation constant (Kd value) of 0.1 nmol/l and a density of 300 fmol/mg of protein. The binding sites are stereoselective and discriminate between (+)-nitrendipine and (-)-nitrendipine. We conclude that a high-affinity binding site at which 1,4-dihydropyridines bind, has now been identified with radioligand binding techniques. This site may well represent the locus where the potent 1,4-dihydropyridines exert their pharmacological action.

Animals↗
PubMed1980

Sodium ions increase the affinity of clonidine for alpha 1-adrenoceptors in rat brain.

alpha 1-Adrenoceptors in rat brain membranes, labeled by the antagonist [3H] prazosin, increase their affinity for clonidine several-fold when sodium ions are added. The conversion of alpha 1-adrenoceptors, buy sodium ion, into a conformation, which has higher affinity for the agonist clonidine, indicates that these receptors as the alpha 2-adrenoceptors are modulated by cations in vitro.

Animals↗